Microscopy of biofilms

Abstract

Identification, visualization and investigation of biofouling microbes are not possible without light, epifluorescence and electron microscopy. The first section of this chapter presents methods of quantification of microbes in biofilms and Catalyzed Reporter Deposition Fluorescent in situ hybridization (CARD-FISH). The second section provides an overview of Laser Scanning Confocal Microscopy (LSCM) imaging, which focuses mainly on the Fluorescent in situ Hybridization Technique (FISH) technique. This technique is very useful for visualization and quantification of different groups of microorganisms. The third section describes the principles of transmission (TEM) and scanning (SEM) electron microscopy. Traditional light and epifluorescent microscopy: Light microscopy is among the oldest methods used to investigate microorganisms. Several light microscopy techniques, such as bright field, dark field and phase contrast, enhance contrast between microorganisms and background. Epifluorescent microscopy is preferable over scanning electron microscopy (SEM) for bacterial size and abundance studies. Fluorescent in situ hybridization (FISH) allows quick phylogenetic identification (phylogenic staining) of microorganisms in environmental samples without the need to cultivate them or to amplify their genes using the polymerase chain reaction (PCR). This method is based on the identification of microorganisms using short (15-20 nucleotides) rRNA-complementary fluorescently labeled oligonucleotide probes (species, genes or group specific) that penetrate microbial cells, bind to RNA and emit visible light when illuminated with UV light. The chapter also lists the materials and equipment necessary for counting bacteria in biofilms using 4',6-diamidino-2-phenylindole (DAPI) staining and catalyzed reporter deposition (CARD)-FISH. Confocal laser scanning microscopy: Laser scanning confocal microscopy (LSCM) imaging offers many advantages over conventional light and fluorescence microscopy, including the elimination of out-of-focus signal and the capability to collect images from serial sections of thick specimens. In situ hybridization with end-labeled oligonucleotide probes fluorescent in situ hybridization (FISH) and amplification of signal from those end-labeled probes catalyzed reporter deposition (CARD)-FISH are detailed in this chapter. LSCM systems are becoming increasingly common in multiuser equipment facilities in research institutions; the methods and sample preparation described are a summary of basic methods used in for determining the phylogenetic makeup of cells in fixed or live biofilms. There is an option to export the files into compatible file formats from image acquisition software, including jpg or tiff, which can then be annotated and saved in a separate location from the raw image files. Electron microscopy: This chapter describes the principal methods for biofilm preparation for Transmission electron microscopy (TEM) or Scanning electron microscopy (SEM) observations. TEM allows identification of the types of microorganisms present in biofilms because its high resolution enables the visualization of organelles specific to various types of microorganisms. Sample preparation for TEM is time consuming and requires highly skilled technicians or investigators experienced in thin sectioning. Investigators using core TEM facilities, however, are usually responsible for fixation of the sample. These investigators should also have an understanding of the processing steps following fixation, should troubleshooting become necessary. SEM and TEM are complementary in biofilm high-resolution characterization because TEM provides a two-dimensional image of intracellular organelles while SEM yields a three-dimensional rendering of the surface of the biofilm, thereby revealing the overall shape of the organisms composing the biofilm as well as their organization relative to each other and to the extracellular matrix.

Publication Title

Biofouling Methods

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